Melanocyte stem cells in the skin: Origin, biological characteristics, homeostatic maintenance and therapeutic potential

Abstract Melanocyte stem cells (MSCs), melanocyte lineage‐specific skin stem cells derived from the neural crest, are observed in the mammalian hair follicle, the epidermis or the sweat gland. MSCs differentiate into mature melanin‐producing melanocytes, which confer skin and hair pigmentation and uphold vital skin functions. In controlling and coordinating the homeostasis, repair and regeneration of skin tissue, MSCs play a vital role. Decreased numbers or impaired functions of MSCs are closely associated with the development and therapy of many skin conditions, such as hair graying, vitiligo, wound healing and melanoma. With the advancement of stem cell technology, the relevant features of MSCs have been further elaborated. In this review, we provide an exhaustive overview of cutaneous MSCs and highlight the latest advances in MSC research. A better understanding of the biological characteristics and micro‐environmental regulatory mechanisms of MSCs will help to improve clinical applications in regenerative medicine, skin pigmentation disorders and cancer therapy. Key points This review provides a concise summary of the origin, biological characteristics, homeostatic maintenance and therapeutic potential of cutaneous MSCs. The role and potential application value of MSCs in skin pigmentation disorders are discussed. The significance of single‐cell RNA sequencing, CRISPR‐Cas9 technology and practical models in MSCs research is highlighted.


INTRODUCTION
Melanocyte stem cells (MSCs) are skin stem cells derived from vertebrate neural crests. 1 During embryonic development, neural crest cells cross the pathway between the somite and the non-neural ectoderm (dorsolateral pathway) to differentiate into melanoblasts. 2Melanoblasts then undergo an extraordinary migration and eventually populate the epidermis and the developing hair follicle (HF). 2 In the basal layer of the epidermis, melanoblasts differentiate into melanocyte (MC) precursor cells, which are intermediate-state cells that have not fully developed into functional MCs.In the HF, melanoblasts differentiate into MC precursor cells in the hair bulb and MSCs in the bulge and hair germ (HG) area. 3MSCs are located in a microenvironment known as a 'niche' with the bulge, which contains multiple stem cells and supporting cells that provide signalling and scaffolding to stem cells. 4oth MC precursor cells and MSCs can differentiate into mature MCs that produce melanin, which are essential for maintaining hair and skin pigmentation and vital skin functions. 5ith the identification of specific lineage markers and advances in stem cell technology, the role of MSCs in the skin is gradually being revealed.The homeostatic maintenance of MSCs is critical for coordinating skin homeostasis, repair and regeneration.More notably, MSCs are closely linked to several skin disorders, including hair graying, vitiligo, wound healing and melanoma.Recognising and understanding the unique biological properties and stem cell-niche interactions of MSCs and their role in skin disease development will pave the way for innovative treatments for a variety of clinical conditions.Unfortunately, there is no available review to refresh the latest advances in MSCs.
Therefore, this review summarises and updates the latest research progress on MSCs, reviews the niche signals that regulate MSCs homeostasis in the HF, and, crucially, discusses the potential therapeutic applications of MSCs in skin pigmentation disorders.The review offers us fresh insights into regenerative medicine, skin pigmentation diseases and skin cancer therapy.

Development and localisation of melanocytic lineage
The melanocytic lineage in vertebrates arises from the trunk neural crest. 1 Between embryonic day 8.5 (E8.5) and E10, transient expression of Kit ligands induces neural crest cells' migration through the dorsolateral pathway to generate melanoblasts.Subsequently, melanoblasts enter the dermis.At around E13.5, they transmigrate from the developing dermis to the epidermis.From E14.5 onward, melanoblasts migrate from the basal layer of the epidermis towards the developing HF, eventually colonising the epidermis and HF and forming different spatially distributed populations, such as MSCs, follicular MCs and epidermal MCs. 2,6,7In fact, MCs have a dual neural crest origin and, in addition to the above-mentioned source, may also originate from Schwann cell precursors, which exploit the ventral migration pathway. 8,9he HF is a functional appendage of the skin that controls hair growth. 10The HF is divided into two compartments: the permanent portion and the transient portion.
The permanent portion, the upper part of the HF, includes the infundibulum and the isthmus.The outer hair root sheath (ORS) at the end of the isthmus protrudes outward to form the follicular bulge, which is believed to be the main habitat of hair follicle stem cells (HFSCs). 11he permanent portion remains stable and generally does not undergo apoptosis or regeneration during HF morphogenesis.The transient portion, the lower part of the HF, shows morphological changes in growth, recession and quiescence during the hair cycle (Figure 1). 12In synchrony with the hair cycle, MSCs in the HF undergo cyclic activation, regression and quiescence phases.During the anagen, quiescent MSCs are activated and migrate from the bulge down to the HF bulb to differentiate into mature MCs.At catagen, differentiated MCs are depleted by apoptosis, although quiescent MSCs remain within the bulge.Upon entry into telogen, MSCs stay stationary in the bulge and HG region (Figure 1). 13,14Distinguishing from previous studies that suggested that MSCs are mostly distributed in the bulge, Sun et al. found that during the telogen, MSCs are mainly distributed in the HG region and a few in the follicular bulge (Figure 1). 3 During the early growth phase, MSCs in the HG are activated and differentiate into transit-amplifying cells with migratory capacity in the intermediate differentiation state.Transitamplifying cells partly migrate to the follicular bulb to differentiate into mature MCs and partly migrate upward to the bulge and exhibit self-renewal capacity.Due to the lack of differentiation signals within the bulge, the majority of transit-amplifying cells can de-differentiate to revert to the stem cell state and return to the HG through the subsequent telogen, thus being used in the next hair cycle. 3his pioneering finding provides important proof of the plasticity of MSCs in physiological conditions.
Interestingly, in adult mouse skin, MSCs are detected in the HF, while in humans, MSCs are also present on the epidermal basement membrane (Figure 2). 15,16This might be due to selective expression in different species during the evolutionary process.In addition to HF and the   2). 17,18Moreover, it has been reported that human subcutaneous adipose tissue harbours MC progenitor cells, which may have the potential to differentiate into mature MCs. 19In the future, further studies are necessary to prove whether MSCs exist in them.In conclusion, these MSCs outside the HF not only offer direct proof for the occurrence of MSCs in the extrafollicular dermis but also provide pigment cell sources for the repigmentation of depigmented diseases in certain hairless areas.

Biological characteristics of MSCs
MSCs are small, oval-shaped cells that have a limited rate of proliferation and lack melanin granules. 20MSCs have unique molecular characteristics, and low expression of housekeeping genes and pigment-related genes is one of the notable features of MSCs.The reported markers of MSCs are summarised in Table 1, including DCT, 21,22 PAX3, 23,24 MITF, 24 SOX10, 21 c-Kit, 25,26 PMEL17, 25 FRIZZLED4, 27 FRIZZLED7, 27 CD34 28 and TFAP2B. 29his significant discrepancy in gene expression strongly demonstrates that MSCs are in an immature state.The 5-bromo-2-deoxyuridine tracking experiment further confirmed that MSCs at the bulge are undifferentiated, cells that are only activated during early anagen, have the capacity to self-renew, and provide differentiated MCs to the HF. 30 Joshi et al. identified follicular MSCs in the telogen into two subpopulations with distinct functional and regenerative properties for the first time by using CD34. 28he subpopulation located in the bulge is predominantly CD34 + -MSCs with characteristics consistent with neural crest stem cells, expressing high levels of NGFR; in comparison to CD34 + -MSCs in the bulge, the CD34 --MSCs subpopulation located in HG is more dominant in MCs differentiation gene expression levels and could induce MCs replenishment more efficiently. 31This fact indicates that MSCs still retain their inherent flexibility or sensitivity to cellular reprogramming.The functional heterogeneity of subpopulations of MSCs makes the study of MC differentiation more challenging.
Unlike other adult stem cells, which usually exhibit unidirectional differentiation, MSCs can transform between stem cells and transit-amplifying cells, which is characterised by 'reversibility'. 3When the MSCs return to HG after completing the HF growth process, they can dedifferentiate to return to the stem cell state, waiting for the next hair growth cycle.
Furthermore, another noteworthy property of MSCs, especially when compared to other stem cell systems, is their isolation.The majority of the time, an HF only houses one MSC, and even when it does, each MSC exists independently of the others. 30,32In fact, there are only a few insights into the molecular characteristics of MSCs, especially for human MSCs, due to their scarcity and the difficulty of sample collection.This generates uncertainty in clinical practice and limits our understanding of pigmentary diseases.

Functions of MSCs
MSCs are the cornerstone for maintaining hair and skin pigmentation as well as vital skin functions.MSCs eventually differentiate into mature MCs in the hair cycle that produce melanin and supply it to the hair shaft and are considered to function as a reservoir of MCs for hair pigmentary units. 15,25Furthermore, MSCs are a major source of epidermal MCs.UV irradiation or Kit ligand expression stimulates MSCs from HF bulge to migrate upward along the ORS to the epidermis and differen-tiate into mature MCs in the mouse. 25,33In addition to being responsible for pigmenting the hair and skin in physiological conditions, MSCs are also involved in repigmentation after various stresses and injuries.These properties of MSCs may be beneficial in the clinical treatment of depigmentation disorders such as vitiligo, a depigmented skin disorder caused by the absence or dysfunction of epidermal MCs. 34MSCs in the HF in vitiligo are activated by UV irradiation or drugs, migrate to the epidermis, proliferate and differentiate into epidermal MCs, which secrete melanin and promote repigmentation of the white macules. 25,35This pattern of repigmentation, which starts at the orifices of the HF, is called the 'perifollicular repigmentation pattern'. 36Subsequently, the discovery of the 'medium-sized spot repigmentation pattern' indicates that MSCs in the dermis and exocrine sweat glands can also develop into epidermal MCs and participate in vitiligo repigmentation. 37,38The various repigmentation patterns that occur are mainly determined by the different sources of residual melanocytic precursor cells, or MSCs, in the epidermis or HF.Obviously, the repigmentation of vitiligo provides an excellent model for investigating MSCs differentiation and function.
The above studies support the notion that MSCs can provide differentiated MCs for hair and epidermis under various physiological and pathological conditions and are crucial for preserving skin function.

REGULATORY MECHANISMS FOR MAINTAINING MSCS HOMEOSTASIS IN THE FOLLICULAR BULGE
0][41] MSCs lack multiple regulators that sustain the growth and development of the melanocytic lineage, but they still maintain homeostasis and play paramount roles in vivo.This is due to the ability of the niche in which MSCs reside to maintain the homeostasis of MSCs as well as promote the quiescence of MSCs to prevent their overgrowth. 23In this section, we discuss the key signalling pathways in the HF niche that maintain homeostasis in MSCs, including Notch, Wnt, TGFB, NFIB, EDNRB and CXCL12 signalling pathways (Figure 3).

Notch signalling
The Notch signalling plays an indispensable role in the maintenance of MSCs in the HF. 42Notch interacts with its ligand to form the intracellular domain of Notch, which translocates to the nucleus to generate a transactivation complex with the transcription factor RBPJ to activate the transcription of downstream target genes like HES1. 43,44argeted Notch1 and Notch2 depletion results in the progressive loss of follicular MSCs with ageing and causes hair graying. 42Targeted deletion of the downstream transcription factor RBPJ results in similar hair graying by inducing apoptosis of melanoblasts and MSCs in the HF. 45HES1 could suppress the initiation of apoptosis and preserve the survival of melanoblasts and MSCs by blocking the expression of genes required for melanoblast apoptosis. 45n addition, the absence of RBPJ also promotes premature maturation of MSCs to generate MCs in the lower part of the permanent portion of the HF. 46In short, the Notch signalling pathway maintains the homeostasis of melanoblasts and MSCs by inhibiting apoptosis and prevents MSCs from differentiating before reaching the hair bulb.

Wnt/β-catenin signalling
Wnt signalling plays an essential role in the maintenance of follicular MSCs.Wnt suppresses the degradation of βcatenin in MSCs cytoplasm by binding to FZD and LRP5/6 receptors. 47Accumulated β-catenin is translocated to the nucleus, where it interacts with the T-cell specific factor/lymphoid enhancer binding factor 1 transcription factor to bind to DNA to regulate the expression of downstream genes (e.g., TYR, DCT, etc.), promoting the proliferation and differentiation of MSCs into mature MCs. 47,48evertheless, Wnt/β-catenin signalling is not activated spontaneously by MSCs, and this process requires the assistance of other cells, such as HFSCs in the bulge or keratinocytes in the hair matrix. 49,50Interestingly, Wnt/βcatenin signalling is only active during the anagen, and it is turned off for the rest of the hair cycle. 48The inhibition of Wnt signalling is necessary to maintain MSCs in an undifferentiated state.High levels of Wnt inhibitors such as DAB2, DKK3 and SFRP1 are expressed in the HF niche, while MSCs themselves express WIF1, DKK5, DAB2 and SFRP1. 49,51These inhibitors inactivate Wnt/β-catenin signalling and keep MSCs in an immature state.Downregulation of Wnt signalling promotes the de-differentiation of MSCs, thus contributing to the maintenance of MSCs. 3 In addition, downstream targets of the Wnt signalling, especially MITF, PAX3 and SOX10, are implicated in the maintenance of MSCs in the niche. 52Collectively, these results indicate that inhibition of the Wnt signalling contributes to the maintenance of undifferentiated MSCs.Meanwhile, the importance of neighboring HFSCs in maintaining the immature state of MSCs was revealed.

TGFB signalling
TGFB signalling performed by HFSCs maintains the quiescence and homeostasis of MSCs by directly regulating the expression of cyclin and differentiation-related genes. 53GFB expression is decreased when MSCs are activated at the early anagen and increased when MSCs enter telogen. 53TGFB binds to its receptor and phosphorylates the downstream effector SMAD2, which not only induces the morphological transition of MCs to MSCs but also downregulates the expression of MITF, PAX3, and downstream melanogenic genes, so as to maintain the quiescence of MSCs and cause cell cycle arrest of MCs. 53,54Deficiency of the TGFBR2 leads to abnormal proliferation and differentiation of MSCs. 53These findings strongly imply that TGFB signalling plays a dual role in the maintenance of MSCs by inhibiting MSCs differentiation and inducing MSCs quiescence.However, the above-described functions are dependent on the presence of BCL2.BCL2, a major regulator of apoptosis, serves an important anti-apoptotic role in the maintenance and survival of MSCs. 53When BCL2 is specifically deficient, TGFB signalling induces apoptosis in MSCs. 53FSCs highly express COL17A1, a hemidesmosomal transmembrane protein attached to the epidermal basement membrane. 55COL17A1 provides a framework structure for HF bulge to maintain the settlement of HFSCs and MSCs, and its defect leads to reduced anchoring of HFSCs and MSCs and atrophy of HF. 56 Notably, COL17A1 deficiency not only induces premature differentiation of MSCs but also promotes aberrant proliferation and differentiation of HFSCs, gradually losing their characteristics, such as impaired TGFB secretion, and then leading to the loss of MSCs. 53This suggests that COL17A1 is required for MSCs' maintenance by regulating TGFB expression.

NFIB signalling
NFIB is a transcription factor associated with cell development, cell regulation and stem cell maintenance. 57As the coordinator of behaviour between HFSCs and MSCs, NFIB has an important function in maintaining the homeostasis of MSCs. 58The conditional knockout of NFIB in HFSCs promotes the premature differentiation of MSCs and the uncoupling of MSCs and HFSCs, indicating that NFIB may be involved in the regulation of MSCs' homeostasis. 58ntrinsically, NFIB serves as a gatekeeper to maintain the homeostasis of stem cells in the HF, and its loss enhances the self-renewal of MSCs as well as disrupts epithelial-MSC synchronisation.Furthermore, EDN2 is identified as a target of NFIB.By targeting the inhibition of EDN2, NFIB considerably prevents excessive proliferation and precocious differentiation of MSCs. 58

EDNRB signalling
EDNRB, a G protein-coupled receptor, is crucial for maintaining the proliferation and differentiation of MSCs. 59onditional knockout of EDNRB resulted in a decrease in the absolute number of MSCs at the bulge and hair graying in mice, demonstrating the necessity of EDNRB for the maintenance of MSCs' homeostasis and proliferation. 60In addition, activation of Wnt signalling or the increase of EDN1/2 could promote the proliferation and differentiation of MSCs by binding to EDNRB. 48

CXCL12 signalling
CXCL12 is a chemokine secreted by HFSCs to modulate cell migration. 61CXCL12 suppresses MSCs differentiation by binding to CXCR7 on the surface of MSCs, dramatically lowering MITF mRNA expression that regulates MCs development and differentiation, and reducing nuclear translocation of β-catenin. 62Besides, high CXCL12 expression competitively inhibits stem cell factor (SCF), thus preventing MSCs from migrating out of the bulge. 62Taken as a whole, CXCL12 attracts and targets MSCs in the proper position and facilitates the maintenance of MSCs in an undifferentiated state.Recently, Wilson et al. reported that BMI1 is an essential molecule for maintaining the stemness of MSCs. 63MI1 is a key molecule that regulates the self-renewal of multiple adult stem cells, and its deletion leads to the progressive depletion of MSCs. 63These studies show that the crosstalk between follicular MSCs and their niche microenvironment plays an important regulatory role in the quiescent maintenance, proliferative differentiation and regenerative capacity of MSCs.HFSCs can precisely regulate the quiescent maintenance and proliferation differentiation of MSCs through specific molecular signals.In addition, since the follicular bulge is positioned in a complex microenvironment, its surrounding signals, such as keratinocytes, neurons, dermal tissues and adipose tissues, may modulate the homeostasis of MSCs. 64,65For instance, the latest study revealed that SPRY1 protein loss in epidermal keratinocytes promotes MSCs migration to the epidermis in a p53/SCF/c-Kit-dependent manner, enriching the understanding of MSCs regulation by keratinocytes. 66

Hair graying
Hair graying is an obvious ageing-associated phenotype.
During the hair cycle, the reduction or absence of melanin synthesised by MCs in newborn hairs is considered to be responsible for gray hair formation. 67Therefore, as the source of MC replenishment, the role of MSCs in hair graying is of greater concern.Earlier, Nishimura et al. attributed the mechanism of age-associated hair graying in mice and humans to incomplete maintenance of MSCs within the HF bulge. 68With physiological age, the number of MSCs in the bulge decreases or disappears, and ectopically differentiated MCs (EPM) emerge.MSCs lose their self-renewal ability and fail to supply MCs for the next hair cycle, subsequently resulting in gray hair (Figure 4B).In addition to physiological ageing, environmental insults and stress also induce MSCs depletion and hair graying.MSCs abrogation in mice can be observed in gray hair caused by ionising radiation or DNA-damaging drugs. 69,70For example, UVB irradiation induces Wnt7a secretion by mouse keratinocytes and promotes β-catenin translocation into the nucleus.Activation of Wnt/β-catenin signalling promotes MSCs differentiation, depletion and premature hair graying. 48However, unlike other stem cells that undergo apoptosis or senescence after DNA damage, this DNA damage response triggers MSCs to form EPM in the niche, which results in hair graying due to the defect in the self-maintenance of MSCs (Figure 4B). 71The find- ing suggested that physiological gray hair may be related to the accumulation of DNA damage that occurs as people age. 71,72In addition, Zhang et al. reported that acute stress promotes the release of norepinephrine from sympathetic nerve terminals innervating the HF bulge, which drives the rapid proliferation and differentiation of quiescent MSCs by binding to the ADRB2 on the surface of MSCs, leading to permanent depletion of MSCs at the HF, which is considered to be the main reason of hair graying. 64This seems to suggest that MSCs depletion linked to long-term accumulated stress may be the cause of physiological gray hair.Ex vivo cultured human follicular MSCs exposed to certain stress signalling mediators, such as ionising radiation, noradrenaline and hydrogen peroxide, exhibited ectopic pigmentation within the bulge similar to that observed in mouse models. 73This study supported the hypothesis that premature ectopic differentiation of MSCs is the key fate of human hair graying.Recently, Sun et al. pioneered the dis-covery that MSCs are retained in the follicular bulge during HF ageing, and their inability to de-differentiate back to the HG is an important contributor to hair graying, suggesting that modulation of the motility of MSCs may be a new approach to preventing hair graying. 3lthough there are many hypotheses about the mechanism of human hair graying, as the source of the HF pigment cycle, once the self-renewal potential of MSCs is disrupted, the downstream melanocytic lineage will be irreversibly and completely lost.As a result, preventing MSCs depletion, suppressing MSCs ectopic differentiation and regulating the MSCs niche might be innovative avenues to prevent or reverse human hair graying.

Vitiligo
Vitiligo is a depigmented skin disorder caused by the destruction of epidermal MCs from a variety of aetiologies. 74Its typical clinical manifestation is progressive hypopigmentation of the skin.The pathological phenotype of vitiligo shows an eventual complete loss of MCs in the epidermis of vitiligo lesions. 75However, surprisingly, complete MSCs remain within the HF of the lesion areas and are similar to normal skin in number and distribution (Figure 4C). 76The appearance of black hairs in vitiligo lesions also confirms the existence of MSCs in the bulge, the reasoning behind which may be related to immune amnesty at the bulge site. 77,78Song et al. recently showed that ADRB2 expression was significantly elevated in the ORS of vitiligo patients, suggesting that norepinephrine binding to ADRB2 may be involved in vitiligo-associated gray hair formation by inhibiting the differentiation of follicular MSCs to mature MCs. 79lthough MSCs may not be directly involved in vitiligo development, several studies have indicated that MSCs are the key and root cause of vitiligo repigmentation.Narrowband UVB (NB-UVB) irradiation induces strong expression of epidermal Wnt7a, which promotes follicular MSCs to differentiate into mature MCs through activation of β-catenin and migrate to the epidermis to complete repigmentation in mice. 33An elegant study showed that proper microinjury promotes the upward migration of quiescent MSCs in the mouse HF to repopulate epidermal MCs by activating the Wnt/β-catenin signalling, promoting vitiligo repigmentation (Figure 4C). 80It was previously considered that vitiligo in hairless areas such as the palmoplantar and mucous membranes was difficult to recover.The 'medium-sized spot repigmentation pattern' revealed, however, that MSCs also exist in small sweat glands and renew themselves under stress, producing progeny MCs for epidermal repigmentation (Figure 4C). 38urthermore, intervening in MSC-associated signalling pathways may promote vitiligo repigmentation.For example, Wnt/β-catenin signalling not only prevents the progression of active vitiligo but also promotes white patch pigmentation. 81These results suggest that drugs or compounds with the function of activating Wnt/βcatenin signalling may be potential candidates for the development of new therapeutic agents for vitiligo.Furthermore, other skin cells neighbouring MSCs, including keratinocytes, fibroblasts and endothelial cells, may play an important role in pigmentation by regulating MSCs. 82For example, UV irradiation may promote the secretion of cytokines and paracrine growth factors (e.g., α-melanocyte-stimulating hormone [α-MSH], endothelin-1, etc.) by keratinocytes through the activation of p53, which may in turn regulate the migration, proliferation, and differentiation of MSCs, thus contributing to the repigmentation of vitiligo. 82,83The binding of α-MSH to the melanocortin-1 receptor (MC1R) is an important signal for melanogenesis.][86] Phase II clinical studies evaluating the pigmentation and safety of subcutaneous bioresorbable Afamelanotide implants for the treatment of facial vitiligo are currently underway (NCT05210582).Furthermore, clinical trials of non-cultured ORS HF cell suspension transplantation containing MSCs have shown favorable skin pigmentation in patients with stable vitiligo (NCT01923142). 87,88These findings show great promise for the application of MSCs in vitiligo clinical treatment.
Collectively, the number, self-renewal ability and niche microenvironment of MSCs are the pivotal factors affecting vitiligo repigmentation.Intervention of aberrant stem cell-associated regulatory factors or signals, reconstruction of the niche microenvironment, or remodelling of the crosstalk of MSCs and HFSCs would be a milestone advancement for the biological therapy of vitiligo patients.

Skin wound healing
Wound healing is a comprehensive and highly coordinated procedure. 89Epidermal wound healing is mainly dependent on the proliferation and differentiation of various skin stem cells, which supply functional cells for tissue regeneration. 90In particular, stem cells at the bulge are considered to represent the ultimate source of the skin epithelial cell lineage for their potential to reconstruct the HF and epidermis. 12Chou et al. demonstrated that under UVB-induced skin wounding, mouse follicular MSCs migrated directly to the epidermis via the MC1R-ACTH-α-MSH pathway to differentiate into mature epidermal MCs to provide a pigmented protective barrier and promote wound repair. 91Although this direct migration pattern led to a decrease in the number of MSCs in the niche, interestingly, the appearance of gray hair was not observed. 91This indicates that the self-maintenance of MSCs is prioritised over the differentiation of MSCs (Figure 4D).However, in contrast to follicular MSCs at wound sites, which preferentially differentiate early in wound healing to maintain skin integrity, at non-wound sites, MSCs appear to function only during the later phases of wound healing. 92This is probably a protective mechanism.In the early phases of wound healing, quiescence of the bulge region at uninjured sites is important to preserve the regenerative potential of the HF, which might otherwise be depleted by the regenerative potential of stem cells within the bulge after wound healing.Simultaneously, this greatly prevents the tumorigenesis induced by the injury.Activation of β-catenin induces MSCs to proliferate and differentiate into epidermal MCs to contribute to epidermal pigmentation during the skin wound healing phase in the mouse. 48These findings demonstrate the incredibly important role played by MSCs in skin wound healing and provide new perspectives for the clinical management of post-traumatic pigment disorders.

Melanoma
Melanoma is a highly aggressive malignancy in the lineage of MCs, and its origin and pathogenesis have not been fully elucidated.Due to their longevity, MSCs accumulate enormous levels of DNA damage during senescence, making them the most likely source of melanoma stem cells. 93In the Tyr-CreER:Braf:Pten mouse model, Moon et al. demonstrated that UVB induces the activation and translocation of MSCs in the HF in an inflammatory and dose-dependent manner, promoting melanogenesis. 94Nevertheless, since Tyr-CreER targets both MSCs in the HF and MCs in the epidermis, it is intrinsically difficult to determine the true origin of melanoma using Tyr-CreER.glands are the origin of melanogenesis but also mimicking the formation of human acral melanoma. 18Stimulated by UV irradiation or Wnt or EDN secreted by the epidermal microenvironment increased the susceptibility of MSCs to BRAF-V600E transformation, which promoted melanomagenesis in the mouse. 98,99hile these findings confirmed that MSCs are the cellular origin of melanoma, surprisingly, human melanoma is uncommon in HFs with rich MSCs.Indeed, melanoma does not grow in quiescent MSCs in the HF bulge, but it does develop once MSCs are activated and move out of the bulge (Figure 4E). 94,100Similarly, in response to stimulation by melanoma risk factors, quiescent MSCs in exocrine sweat glands undergo self-renewal and provide progeny cells to the epidermis through the ducts, resulting in epidermal melanoma. 18This explains the characteristic of a dermoscopy in a 'parallel ridge pattern' of acral melanoma. 38Crucially, these phenomena suggest that the niche in which MSCs reside maintains the quiescence of MSCs and has an inhibitory effect on melanogenesis. 98n recent years, melanoma patients have developed acquired resistance to targeted therapies and immunotherapy, and new therapeutic options are urgently needed.Any factor that affects the development of MSCs and their subsequent processes has the potential to modulate melanogenesis.As a result, a better understanding of the development of MSCs, the regulation of melanogenesis inhibition by the niche in which MSCs reside, and the exogenous stimulation pathways that activate MSCs might open new possibilities for humanity to reconsider the origin and nature of melanoma and design efficient clinical treatment strategies.

TECHNOLOGICAL ADVANCES IN MSCS RESEARCH
Lineage tracking is one of the key components of MSCs research.Compared with tissue sections that only show the status of MSCs at a specific moment in time, 3D in vivo imaging can track the migration and differentiation of MSCs in real time, providing an excellent tool for defining the migration pathways of MSCs in vivo. 3Moreover, two-photon microscopy with high depth penetration, low invasiveness and precise spatial point focusing has recently been used for MSCs lineage tracking. 101ingle-cell RNA sequencing (scRNA-seq) has created new opportunities for research in various fields due to its crucial role in the discovery of new cell populations, dynamic observation of developmental processes and revelation of gene regulatory mechanisms in recent years. 102,103tilising 3D imaging and scRNA-seq to track the process of cellular senescence and movement within each HF in real

Human vitiligo skin biopsies Analysis of MSCs, melanoblasts and MCs in human hair follicles and epidermis
Meanwhile, using CRISPR/Cas9 technology, specific disease models involving MSCs can be constructed to explore the molecular mechanisms of diseases and develop potential therapeutic strategies.In the future, it is anticipated that CRISPR/Cas9 technology will greatly improve the treatment of disorders associated with MSCs due to its advantages in precision, stability and simplicity. 106he development of practical models of MSCs has provided researchers with tools to study MSCs in vitro and in vivo (Table 2).These models include genetically engineered mouse models, 18,30,31,33,48,66,91,94,96,97,101 zebrafish models, 107 human vitiligo skin biopsies 22 and human pluripotent stem cell models. 24These models serve as important tools for studying the biological properties, differentiation potential, disease mechanisms and potential therapeutic strategies of MSCs.Therefore, the development and optimisation of MSCs models are of great scientific and clinical value for the development of skin biology and pigmentation diseases.
Additionally, recently, HF structures constructed entirely from human primary cells have been successfully fabricated using 3D bioprinting technology. 108Threedimensional bioprinting technology can create models with complex structures, which lay the foundation for further research on the differentiation process and biological characteristics of MSCs.Meanwhile, 3D bioprinting technology can also be employed to construct disease models such as vitiligo, which can help to study the role of MSCs in the pathogenesis and treatment of the disease.
In summary, 3D in vivo imaging, two-photon microscopy, scRNA-seq, CRISPR/Csa9, 3D bioprinting technology and practical models provide powerful tools for MSCs research, which can help deepen our understanding of MSCs and advance the development of regenerative medicine and therapeutic strategies for skin pigmentation disorders.

CONCLUSION AND PERSPECTIVES
MSCs located in HFs, epidermis or eccrine sweat glands play a determinant role in maintaining hair and skin pigmentation and important skin functions.The intricate interaction between MSCs and niche provides significant views into skin biology development.More importantly, this review provides fresh insights into the prevention and treatment of gray hair, depigmentation disorders, skin wound healing and melanoma through an intensive exploration of MSCs.Since MSCs are long-lived, multipotent and highly manipulable in vivo and in vitro, they may provide a source of stem cells for the treatment of diverse skin pigment disorders. 109However, research in this field is still in its infancy due to technical limitations and challenges with sample collection.Technologies such as genomics studies, scRNA-seq and CRISPR/Csa9 may provide new approaches to tackle these challenges. 110By using humanderived MSCs and gene editing technology, specific disease models can be constructed, which will help better explore the unknown biological features of human MSCs and provide a theoretical foundation for clinically personalised and precise treatment.Exploiting more human-based methodologies will help us to elucidate the uncharted territories of MSCs that have yet to be identified.In addition, the safety, efficacy and side effects of MSCs in clinical treatment need to be further evaluated.
However, despite the great progress that has been made in the study of MSCs, there are still many problems that have not been fully explored.For example, MSCs may be only one important part of the regeneration and differentiation of MCs, while the role of other precursor cells in the melanocytic lineage on the development and differentiation of MCs needs to be further investigated.In depigmentation diseases, how to effectively activate MSCs and make them play an appropriate and lasting role is a tricky problem that needs to be explored in the future.Similar hair graying is observed, but the difference in the repigmentation rate of gray hair between alopecia areata and vitiligo may suggest different pathogenesis and characteristics of MSCs.In addition, whether extrafollicular MSCs can be recruited to the bulge to promote gray hair repigmentation requires further investigation.
As a result, it is of great significance to further elucidate the specific signals and mechanisms of crosstalk among human MSCs and apply scRNA-seq, gene editing technology, spatial transcriptomics and whole-genome sequencing in MSCs field, which will help establish a new paradigm for skin regeneration research.It is believed that in the near future, the repigmentation of skin and hair will become the pioneers of stem cell transformation, providing the theoretical and practical cornerstones for regenerative medicine in the regeneration and repair of various tissues and organs.

A U T H O R C O N T R I B U T I O N S
Luling Huang, Shuli Li and Chunying Li contributed to the conception, design and final approval of the submitted version.Luling Huang and Yuzhi Zuo contributed to manuscript writing.All authors have read and approved the final manuscript.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare they have no conflicts of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article since no new data were created or analysed in this study.

F
I G U R E 1 A schematic drawing of the hair follicle structure and melanocyte stem cells (MSCs) and their progeny in the hair cycle.(A) During the early anagen phase, quiescent MSCs (shown as blue dots) positioned in the follicular bulge are activated, migrate down along the outer root sheath (ORS), and differentiate into mature melanocytes (MCs) (shown in green) in the follicular bulb.(B) At catagen, mature MCs undergo apoptosis along with the follicular transient portion, while quiescent MSCs remain in the bulge.(C) During telogen, MSCs maintain a quiescent state in the bulge and hair germ (HG) until the onset of the next anagen phase.APM, arrector pili muscle; BG, bulge; DP, dermal papilla; HFSCs, hair follicle stem cells; HS, hair shaft; IRS, inner root sheath; P, permanent portion; SG, sebaceous gland; T, transient portion.F I G U R E 2 The distribution area of melanocyte stem cells (MSCs).(A) In the adult mouse hair follicle, MSCs are located in the bulge region.(B) In the human hair follicle, MSCs can be identified not only in the bulge region but also on the basement membrane of the interfollicular epidermis.(C) MSCs are present in the secretory part of the exocrine sweat glands in humans and mice.

F I G U R E 3
Schematic of the key molecules and signalling pathways involved in the survival and maintenance of melanocyte stem cells (MSCs) in the hair follicle niche.Within the niche, hair follicle stem cells (HFSCs), MSCs themselves and the extracellular matrix are involved in the maintenance of MSCs' homeostasis.Notch, Wnt, TGFB, NFIB, EDNRB and CXCL12 signalling pathways are key pathways in the maintenance of homeostasis in MSCs.

F I G U R E 4
The role of melanocyte stem cells (MSCs) in different skin disorders: microstructural diagram.(A) Normal skin.MSCs (shown as blue dots) are located in the hair follicle bulge and exocrine sweat glands, and mature melanocytes (MCs) (shown as brown dots) are located in the hair matrix and epidermis.(B) Hair graying.The number of MSCs within the bulge is reduced or absent.The MSCs form ectopically differentiated melanocytes (shown as purple dots) that fail to provide functional melanocytes for the next hair cycle (shown as a red arrow), leading to the appearance of gray hair.(C) Vitiligo.Vitiligo lesions show a complete loss of MCs within the epidermis.However, intact MSCs are retained, and their number and distribution are similar to those of normal skin.Under the stimulation of certain specific factors, MSCs in hair follicles and exocrine sweat glands are activated to produce progeny MCs that migrate upward to the epidermis to complete the repigmentation of vitiligo (shown as a green arrow).(D) Skin wound healing.Under skin wounding such as UVB irradiation, follicular MSCs undergo self-renewal (shown as an orange arrow), and then migrate to the epidermis to differentiate into functional MCs to provide a protective pigment barrier (shown as a green arrow).(E) Melanoma.Under the stimulation of risk factors, follicular MSCs are activated, migrate out of the bulge region, and differentiate into MCs in the epidermis (shown as a green arrow), further leading to melanogenesis (shown as a black cluster).
This work was supported by the National Key Research and Development Program of China (2021YFA1101003 and 2023YFC2509005), the National Natural Science Foundation of China (nos.82330096, 82222059 and 81930087) and the R&D Project of Pazhou Lab (Huangpu) under grant (2023K0605).
TA B L E 1 94,97Subsequently, Sun et al. constructed a c-Kit-CreER mouse model targeting MSCs only, clearly showing that oncogenic MCs derived from MSCs invade the underlying dermis and form heterogeneous melanoma that closely resembles human melanoma.97In contr to Moon et al., who claimed that UVB is required to elicit melanoma, Sun et al. proved that MSCs can initiate epidermal melanoma during normal hair regeneration.94,97Remarkably,Eshiba et al.
successfully established a DCT-CreER KI; Braf-CA;Ptenfx/fx mouse model with a fate-tracing system for MSCs, demonstrating not only that MSCs from exocrine sweat

TA B L E 2
Practical models that were used for melanocyte stem cells (MSCs) study.